Insulin-like Growth Factor/Insulin-like Growth Factor 1 Receptor (IGF/IGF1R) signaling plays critical roles in regulating cell growth, proliferation and apoptosis. Both IGFs (IGF1 and IGF2) and IGF1R are commonly involved in human cancers, including leukemia. However, little is known about the role of this pathway in normal hematopoiesis and how its activation contributes to leukemogenesis. Cancer development is highly dependent on cellular context, which is determined by intrinsic genetic programs in the affected cells and by their responses to microenvironment stimuli. The long-term objective of this project is to understand the cell type-specific interplay of signaling pathways and oncogenes, which forms the basis for developing novel and more effective targeted cancer therapies and for cancer prevention. Down syndrome acute megakaryoblastic leukemia (DS-AMKL) and a related preleukemic condition called transient leukemia (DS- TL) are used as a model to study this. DS-AMKL is a unique pediatric leukemia characterized by the triad of fetal origin, trisomy 21, and somatic mutations in the hematopoietic transcription factor, GATA1 (leading to production of a shorter variant of GATA1 called GATA1s). By analyzing Gata1s knockin mice and human DS-TL/DS-AMKL cells, it was found that GATA1s leads to hyperproliferation of fetal megakaryocytic progenitors (MPs), but not their adult counterparts. Fetal MPs are highly dependent on IGF/IGF1R signaling for their proliferation and survival, whereas adult MPs are not. GATA1s mutant fetal MPs and leukemic cells are both hypersensitive to inhibition and activation of IGF/IGF1R signaling. Based on these preliminary data, it is hypothesized that GATA1 may serve as a brake to restrict the IGF/IGF1R signaling (engine)-stimulated proliferation of fetal MPs, likely through repression of E2F target genes, whereas GATA1s is defective in this function. Additional hypotheses include: overactive IGF/IGF1R signaling may cooperate with GATA1s in vivo to initiate DS-TL/DS-AMKL; and fetal and adult hematopoiesis may have different requirements for IGF/IGF1R signaling, a feature capitalized upon by certain types of leukemia to drive their malignant transformation. Three specific aims are proposed to test these: (1) To determine the molecular mechanism by which GATA1 controls target genes of IGF/IGF1R signaling (e.g., E2F targets, including Myc) in fetal megakaryocytes; (2) To test whether constitutively active IGF/IGF1R signaling cooperates with GATA1s in vitro and in vivo to enhance the self-renewal proliferation of fetal MPs. This will be done using transgenic approaches to ectopically express IGF2 in mouse fetal liver cells; (3) To establish and to compare the in vivo role of IGF/IGF1R signaling in fetal versus adult stage hematopoiesis, by conditionally knocking out Igf1r at different stages of hematopoiesis. It is expected that these studies will significantly enhance our understanding of the role of IGF/IGF1R signaling in blood development, and of how its activation contributes to leukemia in a cellular context-dependent manner.